KLHL26 Antibody

What is the domain structure of KLHL26 protein and how does it influence antibody selection?

KLHL26 belongs to the Kelch-like (KLHL) gene family and consists of three distinct domains that should be considered when selecting antibodies: a BTB/POZ domain (for homodimerization), a BACK domain (which connects the BTB domain to the Kelch repeats), and six Kelch repeats that form a single topologic fold . When selecting antibodies, researchers should consider which domain they wish to target based on their experimental goals. Antibodies targeting the Kelch repeats may be useful for studying substrate interactions, while those targeting the BTB/POZ domain might be more appropriate for studying CUL3 interactions and dimerization processes .

What is the primary cellular function of KLHL26?

KLHL26 likely functions as a component of the ubiquitin-proteasome system (UPS), specifically as part of E3 ubiquitin ligase complexes through its interaction with Cullin-3 (CUL3) . The protein's BTB and BACK domains likely interact with CUL3, while the Kelch domain recruits specific targets for ubiquitination . In this capacity, KLHL26 appears to play a role in protein turnover and degradation pathways that are essential for normal cardiac development and function. Antibodies against KLHL26 can help elucidate these protein-protein interactions through co-immunoprecipitation experiments and immunofluorescence co-localization studies .

How is KLHL26 expressed during cardiomyocyte differentiation?

RNA-seq analysis performed on H1-ESCs has demonstrated that KLHL26 is expressed during cardiomyocyte differentiation . This temporal expression pattern suggests KLHL26 plays a developmental role in cardiac tissue formation. When designing experiments to study KLHL26 in cardiac development, researchers should consider this expression timeline and select appropriate time points for antibody-based detection. Immunohistochemistry or immunofluorescence using anti-KLHL26 antibodies at different stages of cardiomyocyte differentiation can help map the spatial and temporal expression patterns of this protein .

How can I validate the specificity of a KLHL26 antibody in my experimental system?

A comprehensive validation approach should include multiple methods:

• CRISPR Knockout Validation: Generate KLHL26 knockout cells using CRISPR/Cas9 systems such as the KLHL26 sgRNA CRISPR/Cas9 All-in-One Lentivector set . Compare antibody signal between wild-type and knockout samples via Western blot, immunofluorescence, or flow cytometry.

• Peptide Competition Assay: Pre-incubate the antibody with a purified KLHL26 peptide corresponding to the epitope, then perform immunostaining or Western blotting. The specific signal should be significantly reduced.

• Multiple Antibody Comparison: Use at least two antibodies targeting different epitopes of KLHL26 to confirm consistent localization and expression patterns.

• Transcript Correlation: Correlate protein detection levels with mRNA expression data from qPCR or RNA-seq analyses.

After knockout validation, perform Surveyor assay or Sanger sequencing on at least 20 isolated clones to confirm successful gene editing before proceeding with antibody validation experiments .

What considerations should be made when studying the KLHL26 (p.R237C) variant with antibodies?

The p.R237C variant, located in the BACK domain of KLHL26, presents unique challenges for antibody-based studies:

• Epitope Accessibility: The variant may alter protein conformation, potentially masking or exposing different epitopes. Consider using antibodies targeting regions distant from the mutation site.

• Altered Interactions: This variant exhibits an altered electrostatic surface profile that may decouple the CUL3 interactome . For co-immunoprecipitation studies, compare binding profiles between wild-type and variant KLHL26 using antibodies against both KLHL26 and potential binding partners.

• Subcellular Localization: The variant protein shows association with distended endo(sarco)plasmic reticulum and dysmorphic mitochondria . Use confocal microscopy with KLHL26 antibodies alongside organelle markers to quantify co-localization differences between wild-type and variant proteins.

• Functional Assays: When performing ubiquitination assays, this variant may show decreased CUL3 binding and decreased turnover of targets . Design protocols incorporating both wild-type and variant proteins, using antibodies to detect changes in ubiquitination patterns.

A detailed comparison of wild-type versus KLHL26 (p.R237C) properties in experimental systems:

How can I design proximity ligation assays (PLA) to study KLHL26 interactions with the ubiquitin-proteasome system?

Proximity ligation assays offer a powerful approach to visualize and quantify endogenous protein-protein interactions involving KLHL26:

• Antibody Selection: Choose a validated KLHL26 antibody raised in one species (e.g., rabbit) and antibodies against suspected interaction partners (CUL3, E2 ligases, or substrate proteins) raised in different species (e.g., mouse).

• Control Design: Include necessary controls:

  • Negative controls: Omit one primary antibody

  • Specificity controls: Use KLHL26 knockout cells

  • Positive controls: Known interacting partners

• Optimization Strategy:

  • Begin with standard PLA protocol parameters

  • Adjust antibody concentrations to minimize background

  • Optimize blocking conditions to prevent non-specific binding

  • Test different fixation methods (PFA vs. methanol) as protein conformation affects epitope accessibility

• Quantification Method:

  • Count interaction puncta per cell using image analysis software

  • Compare wild-type KLHL26 versus p.R237C variant interaction profiles

  • Analyze changes in interaction patterns under different cellular conditions (e.g., proteasome inhibition)

The PLA data should be validated with complementary techniques such as co-immunoprecipitation and FRET analysis to confirm the interactions detected .

How can KLHL26 antibodies be used to investigate cardiac pathophysiology in Ebstein's anomaly models?

KLHL26 antibodies are valuable tools for investigating the mechanisms underlying Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC):

• Immunohistochemical Analysis: Use KLHL26 antibodies for comparative studies of cardiac tissue from normal versus EA/LVNC affected samples to examine expression levels and localization patterns. Focus on regions known to be affected in EA, such as the tricuspid valve and right ventricle .

• Sarcomeric Disarray Assessment: Since electron microscopy has revealed disrupted Z-bands in the right atrium and atrialized part of the right ventricle of EA patients, combine KLHL26 antibodies with antibodies against sarcomeric proteins for co-localization studies . This can help determine if KLHL26 colocalizes with disrupted sarcomeric structures.

• iPSC-CM Disease Modeling: In iPSC-derived cardiomyocytes carrying the KLHL26 (p.R237C) variant, use antibodies to track:

  • Altered morphology of endo(sarco)plasmic reticulum and mitochondria

  • Changes in contractile apparatus organization

  • Abnormal calcium handling components

• Developmental Time-Course: Perform immunofluorescence studies at different developmental stages to determine when and where KLHL26 dysfunction impacts cardiomyocyte differentiation, correlating antibody signals with functional measures such as calcium transients and contractility .

What methods can be used to determine if KLHL26 antibodies detect alterations in protein turnover in cardiac disease models?

To investigate if KLHL26-mediated protein turnover is altered in cardiac disease:

• Proteome Analysis:

  • Combine immunoprecipitation using KLHL26 antibodies with mass spectrometry to identify bound substrates

  • Compare substrate profiles between wild-type and p.R237C variant KLHL26

  • Validate key targets with co-immunoprecipitation and Western blotting

• Ubiquitination Assays:

  • Use KLHL26 antibodies to pull down the protein and its complexes

  • Probe with anti-ubiquitin antibodies to assess ubiquitination activity

  • Compare ubiquitination levels of specific target proteins between normal and disease models

• Protein Stability Measurements:

  • Perform cycloheximide chase experiments to measure protein half-lives

  • Use antibodies to detect decay rates of putative KLHL26 substrates

  • Compare turnover rates between wild-type and KLHL26 (p.R237C) expressing cells

• In Vivo Degradation Dynamics:

  • Design pulse-chase experiments with KLHL26 antibody detection

  • Incorporate proteasome inhibitors to confirm UPS-mediated degradation

  • Use fluorescence recovery after photobleaching (FRAP) with fluorescently-tagged targets and correlate with antibody-based quantification

These methodologies can help elucidate how the p.R237C variant might dysregulate the degradation of sarcomeric proteins, potentially explaining the altered cardiomyocyte proliferation and differentiation seen in EA/LVNC cases .

How can KLHL26 antibodies be used to investigate the "structural constituent of muscle" pathway suppression observed in KLHL26 variant models?

RNASeq data from models with the KLHL26 (p.R237C) variant indicated suppression of the "structural constituent of muscle" pathway . Antibody-based approaches to investigate this finding include:

• Protein Expression Profiling:

  • Perform quantitative Western blotting using antibodies against KLHL26 and key muscle structural proteins (e.g., sarcomeric proteins)

  • Compare expression levels between wild-type and variant cells at multiple differentiation timepoints

  • Create correlation matrices between KLHL26 levels and structural protein abundance

• Spatial Organization Analysis:

  • Use super-resolution microscopy with KLHL26 antibodies and muscle structural protein markers

  • Quantify spatial relationships and organizational patterns

  • Detect potential abnormal aggregation or mislocalization of structural proteins

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • If KLHL26 affects transcription factors controlling muscle structural genes, use KLHL26 antibodies for ChIP experiments

  • Identify potential regulatory relationships between KLHL26 and transcription of muscle structural genes

  • Compare ChIP profiles between wild-type and variant models

• Protein-Protein Interaction Network Mapping:

  • Use KLHL26 antibodies for immunoprecipitation followed by mass spectrometry

  • Build interaction networks focused on muscle structural components

  • Identify differences in interaction networks between normal and disease states

These approaches can help determine whether KLHL26 directly regulates muscle structural proteins through ubiquitination or indirectly through other regulatory mechanisms .

What are the optimal fixation and permeabilization conditions for KLHL26 immunofluorescence in different cell types?

Optimizing fixation and permeabilization conditions is critical for KLHL26 antibody performance in immunofluorescence:

• For iPSC-derived Cardiomyocytes:

  • Primary fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Secondary fixation options: Compare with 100% ice-cold methanol for 5 minutes (especially if examining ER/SR structures)

  • Permeabilization: 0.1% Triton X-100 for 10 minutes

  • Additional step: Antigen retrieval methods may improve signal, particularly when examining the KLHL26 (p.R237C) variant with distended ER/SR structures

• For Primary Cardiac Tissue Sections:

  • Fresh frozen sections: Acetone fixation for 10 minutes at -20°C

  • FFPE sections: Standard deparaffinization followed by citrate buffer (pH 6.0) antigen retrieval

  • Permeabilization: 0.2% Triton X-100 for 15 minutes

  • Special consideration: Autofluorescence quenching may be necessary due to lipofuscin in cardiac tissue

• For Standard Cell Lines (HEK293, HeLa, etc.):

  • Fixation: 2% paraformaldehyde for 10 minutes at room temperature

  • Permeabilization: 0.1% Triton X-100 or 0.5% saponin (if gentler permeabilization is needed)

  • Alternative: −20°C methanol for combined fixation/permeabilization when examining KLHL26 association with the cytoskeleton

Perform systematic optimization by testing different combinations of fixation and permeabilization methods, as KLHL26's multi-domain structure may require specific conditions to preserve protein conformation and epitope accessibility .

How can I optimize Western blot protocols for detecting KLHL26 in tissues with varying expression levels?

Optimizing Western blot protocols for KLHL26 detection requires careful consideration of several factors:

• Sample Preparation:

  • For cardiac and muscle tissues (high expression): Standard RIPA buffer with protease inhibitors

  • For tissues with lower expression: Consider using NP-40 buffer with phosphatase inhibitors and gentle homogenization

  • Sonication parameters: 3-5 short pulses (5 seconds each) to preserve protein integrity

• Protein Loading and Transfer:

  • For high-expression tissues: 20-30 μg total protein

  • For low-expression tissues: 50-75 μg total protein

  • Transfer conditions: Semi-dry transfer at 15V for 60 minutes for standard sized KLHL26 (predicted MW ~65 kDa)

  • Membrane selection: PVDF membranes (0.45 μm pore size) provide better protein retention

• Blocking and Antibody Incubation:

  • Blocking: 5% non-fat dry milk in TBST for 1 hour (preferred over BSA for reduced background)

  • Primary antibody: Incubate at 4°C overnight with gentle rocking (1:500-1:1000 dilution range)

  • Secondary antibody: HRP-conjugated, 1:5000-1:10000 for 1 hour at room temperature

  • Consider using signal enhancers for tissues with low expression

• Detection Optimization:

  • For standard detection: ECL substrate

  • For low abundance: High-sensitivity ECL or femto-based substrates

  • Exposure time: Begin with 30 seconds and adjust as needed

  • Consider using a gradient of known positive controls to establish a detection range

Include appropriate loading controls and KLHL26 knockout samples when available to verify antibody specificity .

What strategies can resolve antibody cross-reactivity issues when studying KLHL26 in the context of other KLHL family proteins?

The KLHL protein family contains multiple members with similar domain structures, presenting challenges for antibody specificity:

• Epitope Selection Strategy:

  • Choose antibodies raised against unique regions of KLHL26, avoiding conserved BTB/POZ and Kelch domains shared across the family

  • Target the inter-domain linker regions that show greater sequence divergence

  • When selecting commercial antibodies, request epitope mapping information to assess potential cross-reactivity

• Validation Approaches:

  • Perform parallel Western blots with recombinant KLHL family proteins to identify cross-reactivity

  • Use KLHL26 CRISPR/Cas9 knockout cells as negative controls

  • Include siRNA knockdown of KLHL26 with quantification of signal reduction

  • Test antibodies on tissues with differential expression of KLHL family members

• Experimental Controls:

  • Run comparative blots with antibodies against other KLHL family members

  • Perform peptide competition assays using peptides from KLHL26 and related family members

  • Include expression analysis of multiple KLHL proteins in your experimental system

• Advanced Techniques for Ambiguous Results:

  • Immunoprecipitation-mass spectrometry to confirm antibody targets

  • Two-dimensional gel electrophoresis to separate closely related proteins

  • Develop isoform-specific detection methods combining antibodies with molecular weight discrimination

Creating a cross-reactivity profile table for your specific antibody against the most closely related KLHL family members can help document specificity and guide experimental interpretation .

How can KLHL26 antibodies be used in single-cell proteomics approaches to study heterogeneity in cardiac development?

Single-cell proteomics offers exciting opportunities to explore KLHL26's role in cardiac cell populations:

• Mass Cytometry (CyTOF) Applications:

  • Metal-conjugated KLHL26 antibodies can be incorporated into CyTOF panels

  • Combine with markers for cardiac progenitor populations, cell cycle regulators, and differentiation markers

  • Create high-dimensional maps of KLHL26 expression across cardiac developmental trajectories

  • Compare KLHL26 (p.R237C) variant versus wild-type expression patterns at single-cell resolution

• Imaging Mass Cytometry Protocols:

  • Optimize metal-tagged KLHL26 antibodies for tissue section analysis

  • Develop multiplexed panels to simultaneously visualize KLHL26 with cardiac structural proteins and signaling molecules

  • Quantify spatial relationships between KLHL26-expressing cells and anatomical features relevant to Ebstein's anomaly

• Single-Cell Western Blotting:

  • Adapt protocols for microfluidic single-cell Western blots using KLHL26 antibodies

  • Compare protein levels across individual cells from normal and disease models

  • Correlate with functional cellular phenotypes

• Spatial Transcriptomics Integration:

  • Combine antibody-based protein detection with spatial transcriptomics

  • Create multimodal single-cell atlases integrating KLHL26 protein levels with transcriptional profiles

  • Map developmental trajectories where KLHL26 function is critical

These approaches can help identify specific cardiac cell populations where KLHL26 dysfunction has the greatest impact, potentially revealing therapeutic targets for EA/LVNC .

What considerations should be made when developing phospho-specific antibodies for KLHL26?

While phospho-regulation of KLHL26 is not yet well-characterized, developing phospho-specific antibodies requires careful planning:

• Phosphorylation Site Prediction and Validation:

  • Use computational tools to predict potential phosphorylation sites in KLHL26

  • Prioritize sites near functional domains, especially the BACK domain where the p.R237C variant occurs

  • Validate predicted sites using mass spectrometry of immunoprecipitated KLHL26 from cardiac tissues/cells

  • Focus on sites that may regulate CUL3 binding or substrate recognition

• Antibody Development Strategy:

  • Design phosphopeptide antigens containing the site of interest plus 7-10 flanking amino acids

  • Generate paired antibodies: one phospho-specific and one recognizing the same region regardless of phosphorylation

  • Include a phosphatase treatment control in validation experiments

  • Test specificity across different stimulation conditions that might alter KLHL26 phosphorylation

• Functional Validation Approaches:

  • Correlate phosphorylation with CUL3 binding efficiency

  • Assess impact on substrate ubiquitination using in vitro assays

  • Map phosphorylation dynamics during cardiac development stages

  • Compare phosphorylation patterns between wild-type and KLHL26 (p.R237C) variant

• Experimental Applications:

  • Develop phosphorylation state-specific immuno-enrichment protocols

  • Design assays to screen for kinases and phosphatases regulating KLHL26

  • Map phosphorylation changes in response to cardiac stress conditions

Phospho-specific antibodies could reveal regulatory mechanisms controlling KLHL26's role in the ubiquitin-proteasome pathway and cardiac development .

How can KLHL26 antibodies be incorporated into high-content screening approaches to identify modulators of protein function?

High-content screening with KLHL26 antibodies enables discovery of functional modulators:

• Assay Development Strategy:

  • Design cell-based assays in cardiac relevant cell lines (e.g., iPSC-CMs) expressing wild-type or p.R237C KLHL26

  • Develop multiplexed immunofluorescence protocols with KLHL26 antibodies and markers for:

    • Subcellular localization (especially ER/SR and mitochondria)

    • Ubiquitination activity

    • Sarcomeric organization

    • Calcium handling components

• Screening Parameters:

  • Primary readouts: KLHL26 localization, protein levels, co-localization with CUL3

  • Secondary readouts: Target protein ubiquitination, proteasome activity, sarcomeric organization

  • Tertiary readouts: Cell morphology, calcium transients, contractility

  • Control compounds: Proteasome inhibitors, E1/E2 inhibitors

• Hit Validation Framework:

  • Dose-response relationships for primary hits

  • Orthogonal assays to confirm mechanism of action

  • Counter-screens to eliminate non-specific effects

  • Validation in disease-relevant models (EA/LVNC iPSC-CMs)

• Advanced Applications:

  • CRISPR-based genetic modifier screens combined with KLHL26 antibody readouts

  • Small molecule library screening to identify compounds that restore normal KLHL26 function

  • Development of targeted protein degradation approaches for dysfunctional KLHL26 variants

This approach could identify compounds that correct the decreased contractions, altered calcium transients, and increased proliferation observed in cells with the KLHL26 (p.R237C) variant .